Efficient shRNA-based knockdown of multiple target genes for cell therapy using a chimeric miRNA cluster platform.

Area of Science:

• Biotechnology
• Immunotherapy
• Molecular Biology

Background:

• Genome engineering tools enhance cell-based immunotherapy but face challenges with multiple gene edits.
• CRISPR-based gene editing is a common method, but alternatives are sought for specific applications.

Purpose of the Study:

• To develop a microRNA (miRNA)-based multiplex short hairpin RNA (shRNA) platform for simultaneous, non-editing-based modulation of multiple genes in engineered cells.
• To create a streamlined, one-step process for generating engineered CAR T-cells with multiple functional modifications.

Main Methods:

• A chimeric cluster of highly efficient miRNA scaffolds was engineered to deliver up to four shRNA-like sequences.
• The platform's "plug-and-play" design allowed for easy swapping of shRNA-derived guide sequences.
• The system was integrated with CAR components for a one-step cell engineering process.

Main Results:

• The platform enabled the delivery of up to four shRNA-like sequences for multiplex gene modulation.
• Functional knockout or fine-tuning of target gene expression was achieved without gene editing.
• The system demonstrated easy, safe, efficient, and tunable simultaneous modulation of multiple target genes.

Conclusions:

• The developed miRNA-based multiplex shRNA platform offers an efficient and safe alternative for engineering cell-based immunotherapies.
• This approach facilitates the introduction of multiple transcriptomic modifications, potentially enhancing engineered cell performance in challenging environments like solid tumors.